MHC class II antigen presentation by intestinal epithelial cells fine-tunes bacteria-reactive CD4 T cell responses
MHC class II antigen presentation by intestinal epithelial cells fine-tunes bacteria-reactive CD4 T cell responses
10% control conditioned media for 20h Organoids were cultured as previously described.1 Colonic crypts were isolated from longitudinal opened and cleaned colon tissue of Atg16l1fl/fl mice by 2mM EDTA in PBS incubation for 90min at 4°C. The tissue was vigorously resuspended in PBS/10% FCS and the supernatant containing the crypts filtered through a 100μm cell strainer and pelleted. Crypts were seeded in 20μl Matrigel/well (Corning) in a 24-well plate. After polymerisation of the Matrigel, 400ul of complete organoid medium was added [Advanced Dulbecco's Modified Eagle Medium (DMEM)/F12 supplemented with 1% Pen/Strep, 10mM HEPES, 1X GlutaMax, 50ng/ml mEGF, 1× N2, 1× B27 (all from Invitrogen), 1mM N-acetylcysteine (NAC), 10μM RhoK-inhibitor (Y-27632 dihydrochloride monohydrate) (both from Sigma) and conditioned medium containing Wnt3a (20%), Noggin (20%) or R-spondin (10%)]. The conditioned media were generated from Rspol-Fc Hek 293T cells (kind gift from Dr. Calvin Kuo, Stanford) and Nog-Fc Hek 293T cells (kind gift from Dr. Gjis van den Brink, Amsterdam) and Wnt3A L cells (ATCC CRL-2647). The medium was changed every 2-3d and organoids were expanded over several passages before they were used for experiments with indicated stimuli. Ref: 1 Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009). Qiagen Rneasy Kit Illumina Truseq mRNA kit